PROJECT SUMMARY: Regulation of gene expression is of paramount importance in animal development, while improper regulation results in developmental defects and disease states. In developing tissues, genes are often initiated through long-range signals, called morphogens, and refined/regulated by gene regulatory networks. Recently, the dynamics of morphogen gradients have been found to impact gene regulation. As sig- naling dynamics are often regulated by feedforward loops (FFLs), the authors aim to determine the role of a feedforward loop in gene expression dynamics in early fruit fly (Drosophila melanogaster) embryos, specifically through development of novel methodologies that enable live measurements of protein and mRNA expression. Our long-term goal is to determine how cells interpret signals and make real-time transcriptional decisions. Therefore, our overall objective in this proposal is to develop novel methodologies to measure live mRNA and protein expression in order to measure the effect of a FFL in the early Drosophila embryo. Our central hy- pothesis is that the FFL is essential to stabilize the activity of a dynamic signal. Our hypothesis is based on literature, preliminary data, and the nature of FFLs, which are typically designed to regulate dynamics. If correct, this hypothesis would give insight into the role of feedforward loops in the context of the dynamics of living tissues in a multicellular organism. The rationale for the proposed research is that our knowledge of the relationship between time-variant signaling and transcriptional response is currently weak, limited by the cur- rently available real-time imaging tools. We plan to test our central hypothesis, thereby attaining the objective of this application, by proposing the following two Specific Aims: Specific Aim 1: Establish a protein-binder based platform to measure dynamics of mRNA and newly- synthesized proteins. In the case of the early Drosophila embryo, GFP maturation time precludes studying the earliest patterning stages. The proposed platform will comprise protein-based binders engineered to pro- duce a bright FRET signal upon binding to the protein/RNA, and will not have the common drawbacks of high background, faint signal, and delivery issues. Specific Aim 2: Validate the sensors from Aim 1 using an in vivo FFL system. Measurements in wildtype embryos will validate our novel sensors. The FFL system we chose to study also exhibits rapid dynamics, so measurements of this system with our sensors will help deter- mine the role of the FFL. We will also genetically perturb the activity gradient by both examining mutants and by mutating crucial regulatory DNA sites. Measurements of embryos with genetically perturbed signal dynam- ics or activity will reveal a causative relationship between signaling and stability of gene expression bounda- ries. Thus, it will test the role of the FFL in a quantitative fashion. Finally, we will synthesize our data into a mathematical model of this FFL system. We expect these outcomes to have a positive impact by setting the stage to determine how cells make decisions ? such as to migrate, differentiate, or undergo apoptosis.